A. Borrelia burgdorferi, the agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), nitrogen dioxide (NO2), nitrogen trioxide (N2O3) and peroxynitrite (NO3). There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs during tick feeding and as the bacteria migrate through the tick salivary glands during transmission. In FY 2018, we determined the roles of osmolarity and reactive nitrogen species (RNS) in survival and gene regulation during the infective cycle. B. burgdorferi must adapt to distinctly different environments in its tick vector and various mammalian hosts. Effective colonization (acquisition phase) of a tick requires the bacteria to adapt to tick midgut physiology. Successful transmission (transmission phase) to a mammal requires the bacteria to sense and respond to the midgut environmental cues and up-regulate key virulence factors before transmission to a new host. Our recent data suggests that osmolarity and RNS are such signals. Osmolarity, while constant in the blood, interstitial fluid and tissue of a mammalian host (300 mOsm), osmolarity fluctuates in the midgut of feeding I. scapularis (600-300-600 mOsm). Remarkably, these relatively small changes affect two independent regulatory networks that promote acquisition and long-term survival (Hk1-Rrp1) as well as transmission (Rrp2-RpoN-RpoS) of B. burgdorferi. Most importantly, recent data from our laboratory shows that dicyclic-GMP, produced by Rrp1, stimulates the phosphatase activity of Hk2. We believe this crosstalk is essential for coordinating these two essential regulatory systems. In related studies, we have shown that RNS that are only present in the midgut of feeding ticks, presents a significant challenge to long-term survival of B. burgdorferi. The damage mediated by RNS stimulates the nucleotide excision repair (NER), base excision repair (BER) and mismatch excision repair (MER) systems which ensures maximum growth and long-term survival. Data from Dr. T. Bourrets laboratory at Creighton University suggests that the response to RNS is mediated by DksA and ppGppp (synthesized by RelA). These data suggest that; (1) dicyclic GMP, triggered by osmolarity, might be an important regulatory modulator that coordinates Hk1/Rrp1 and Hk2/Rrp2-dependent regulation, and (2) RNS stimulates DksA-dependent gene regulation that is essential for the long-term survival of B. burgdorferi in ticks (2). B. B. burgdorferi's ability to adapt and survive in very different environments (tick versus mammal) is attributed to its ability to sense changes in temperature, pH, cell density, oxygen, manganese and/or exposure to host factors and alter gene expression accordingly. Previous reports have demonstrated that central to the regulation of these responses are the sigma factors, RpoN and RpoS. Importantly, RpoN-dependent regulation of RpoS is responsible for the expression of key virulence factors (e.g., OspC, OspA and DbpA) required for infectivity and transmission during the infective cycle. The activities of RpoN are tightly controlled and require ATP-dependent activation. Many of the activators of RpoN-RNA polymerase (RNAP) are response regulators (RR) of two-component regulatory systems and phosphorylation of these RR results in their activation. These RRs are phosphorylated by small molecular weight phosphate donors or, more commonly, by their cognate protein histidine kinase (HK) in response to an environmental signal. The activator of RpoN in B. burgdorferi, Rrp2 encoded by bb0763 (rrp2), is also a RR of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, Hk2 encoded by bb0764 (hk2). These regulatory components form the Rrp2-RpoN-RpoS signaling cascade that coordinates the expression of virulence factors required for successful transition of B. burgdorferi from its arthropod vector to mammalian hosts. Activation of Rrp2 is essential to initiate this regulatory pathway. The intracellular signal and phosphorylation processes triggering Rrp2 activation are poorly understood. Published reports have suggested that acetyl-phosphate (AcP), a global regulatory molecule in many bacteria, serves as a signal and high energy phosphate donor for Rrp2 activation. In a FY 2018 study led by GRC staff scientist, Dr. D. Dulebohn, we identified an environmental condition that affects gene expression and long-term survival: acid stress (2). Our study identified an acid stress response in B. burgdorferi that activated the Rrp2-RpoN-RpoS signal transduction cascade, some BosR-directed oxidative stress response genes, and increased the expression of genes encoding proteins involved in restoring pH homeostasis. An increase in the transcription of these genes was stimulated by membrane-permeable monocarboxylic acids, some of which (acetate and lactate) were identified in the tick midgut following feeding. Moreover, we have now demonstrated that perturbations in pH homeostasis activated the Rrp2-RpoN-RpoS/BosR regulatory cascade(s) that was previously attributed to Acetyl-P. We did not identify the intracellular signal that ultimately up-regulates these pathways, however, the effects of the decrease in pHi on the vacuolar ATPase might suggest that ATP or GTP may serve as effective intracellular energy sensors. While it seemed clear that the acid stress response and pHi effected virulence factor expression in B. burgdorferi in vitro, it seemed unlikely that these parameters affect transmission in vivo. Since the expression of virulence factors, like OspC, are so tightly linked to RpoS, in vitro conditions that trigger Rrp2-RpoN-RpoS and BosR-dependent gene regulation don't necessarily indicate that these conditions are required for, or play a role in, successful transmission. Considering the complexity of the tick midgut, it seems likely that multiple factors are required to maximize and synchronize the expression of virulence factors to the tick feeding cycle to promote successful transmission (4).